Project Summary/Abstract Ubiquitin ligases (E3s) represent a diverse and conserved group of enzymes, with over 600 members. These ligases collectively attach the small protein ubiquitin to more than twenty five percent of the proteome, thereby regulating the stability or activity of each target. Despite the importance of this set of enzymes, only a small percentage of ubiquitin ligases have well-characterized biological functions. We have conducted a CRISPR screen examining the sensitivity of mutations of genes encoding human ubiquitin ligases and deubiquitinating enzymes to a panel of inhibitors covering a broad range of biological pathways. From this screen, we identified an F box protein, called FBXO42, whose mutation renders cells sensitive to inhibitors of mitosis and causes the accumulation of cells with mitotic defects. F box proteins are substrate adaptors for the SCF cullin RING ligase. One of the greatest challenges in the study of ubiquitin ligases is identifying their substrates. We developed several methods to accomplish this, using (MS) spectrometry and fluorescent detection. Leah will identify mitotic substrates of FBXO42 using ligase trap fusion proteins and through genetic screens. By identifying FBXO42 substrates, we will be able to better understand its role in the cell cycle.